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The present invention relates to novel monoclonal antibodies reactive with lipid transfer proteins typically found in foaming beverages. More specifically, the present invention relates to novel monoclonal antibodies raised against the native and denatured forms of barley lipid transfer protein 1, and an assay for determining the content of said proteins in foaming beverages at various stages of their production.
Foaming beverages, e.g. beer and some soft drinks, are popular items in today""s marketplace. In addition to taste, the appearance of the beverage and its ability to form a stable head of foam when poured are important characteristics. In beer, the foam head is one of the first characteristics that a consumer generally uses to judge beer quality.
Foam formation and retention are two factors considered when defining foam quality. The rate at which the foam head forms and collapses depends upon, among other things, the composition of the beer.
Several different beer and foam proteins have been suggested to be important in foam formation and head retention. One such protein is the lipid transfer protein found in cereal grains. In particular, several different studies have analyzed the lipid transfer protein from barley and have shown that its presence in beer exhibits a positive effect on foam formation and stability. Evans and Hejgaard, xe2x80x9cThe Impact of Malt Derived Proteins on Beer Foam Quality. Part I: The Effect of Germination and Kilning on the Level of Protein Z4, Protein Z7, and LTP1xe2x80x9d, J. Inst. Brewing, 105:3:159-169 (1999); Evans et al., xe2x80x9cThe Impact of Malt Derived Proteins on Beer Foam Quality. Part I: The Influence of Malt-positive Proteins and Non-starch Polysaccharides on Beer Foam Qualityxe2x80x9d, J. Inst. Brewing, 105:2:171-177 (1999); Lusk et al., xe2x80x9cFoam tower fractionation of beer proteins and bittering acids,xe2x80x9d European Brewery Convention Beer Foam Quality Symposium (Amsterdam, Oct. 25-27, 1998); Bock et al., xe2x80x9cNew Analytical Techniques with Relevance for the Brewing Industryxe2x80x9d, Brygmesteren, 54(5):47-55 (1997); Lusk et al., xe2x80x9cIndependent role of beer proteins, melanoidins and polysaccharides in foam formation,xe2x80x9d J. Am. Soc. Brew. Chem., 53(3):93-103 (1995); Sorenson et al., xe2x80x9cBarley Lipid Tranfer Protein 1 is Involved in Beer Foam Formationxe2x80x9d, MBAA Tech. Quarterly, 30:136-145 (1993).
Two members of the lipid transfer protein gene family are expressed in barley grain, LTP1 and LTP2. Of the two proteins, only LTP1 is found in beer (see, Evans and Hejgaard, supra). LTP1 is an albumin protein primarily expressed in the aleurone layer of the barley seed. It has a molecular weight of 9,694 Daltons and contains 91 amino acid residues, including 8 cysteines. The amino acid sequence of LTP1 is set forth in SEQ ID NO:1. Studies by Bock et al., supra, have shown that LTP1 is modified during the malting and brewing process to a denatured form (fLTP). It is this denatured form that is believed to effect foam formation and stability.
Other studies have suggested that other proteins and polypeptides are important in foam formation and stability. In particular, it has been suggested that beer and foam proteins of a molecular weight greater than 5,000 Dalton tend to be foam-positive, while polypeptides of molecular weights below 5,000 Dalton tend to be foam-negative. For example, studies by Sharpe et al. have suggested that head retention was related to the ratio of high and low molecular weight polypeptides. (Sharpe et al., xe2x80x9cRapid methods of measuring the foam-active nitrogenous components of worts and beersxe2x80x9d, Proc. Eur. Brewing Conv.: 18th Cong., 607-614 (1981)). Meanwhile, Yokoi, et al, has suggested that protein Z, a 40,000 Dalton barley albumin, plays the most significant role in foaming and head retention (Yokoi et al., xe2x80x9cCharacterization of beer proteins responsible for the foam of beerxe2x80x9d, Proc. Eur. Brewing Conv.: 22nd Cong., 503-512 (1989)). On the other hand, Kauffman et al. has suggested that the prolamin storage proteins of barley, called hordeins, are also important in foam formation and stability (Kauffman et al., xe2x80x9cImmunological Characterisation of Barley Polypeptides in Lager Foamxe2x80x9d, J. Sci. Food Agric., 66:345-355 (1994)).
Most of the above conclusions have resulted from investigations generally involving the fractionation of beer proteins and a determination of their foaming effect. More recently, there has been considerable interest in tracing the origin of foam proteins using immunological methods. Polyclonal antibodies against barley, malt, beer and yeast proteins have been developed and used in these studies. For example, Hollemans and Tonies used polyclonal antibodies to remove polypeptides from beer to establish their effect on foaming (Hollemans and Tonies, xe2x80x9cThe role of specific proteins in beer foamxe2x80x9d, Proc. Eur. Brew. Conv.: 22nd Cong., 561-568 (1989)); Ishibashi et al., used polyclonal antibodies to analyze both foam and haze proteins in beer (Ishibashi et al., xe2x80x9cDevelopment of a new method for determining beer foam and haze proteins by using the immunochemical method ELISAxe2x80x9d, J. Am. Soc. Brew. Chem., 54(3):177-18)); and Bech et al. used polyclonal antibodies to determine the concentration of LTP1 in wort, beer, and barley and malt extracts from several different barley varieties (EP 0728188). The information obtained using polyclonal antibodies, however, is partly limited due to problems of polyspecificity resulting from the presence of immunodominant repetitive hordein sequences (Mills et al., xe2x80x9cImmunological Study of Hydrophobic Polypeptides in Beerxe2x80x9d, J. Agric. Food Chem., 46:4475-4483 (1998)). Accordingly, more exact methods for performing immunological studies on beer and foam proteins are needed.
Monoclonal antibodies have been employed in some cases to avoid the problems associated with the use of polyclonal antibodies. For example, Kaufman et al., supra, has reported the use of monoclonal antibodies against wheat prolamins to study hordein-type material found in beer and foam fractions. Sheehan and Skerritt have also used monoclonal antibodies to examine modifications of hordeins during beer production (Sheehan and Skerritt, xe2x80x9cIdentification and Characterisation of Beer Polypeptides Derived from Barley Hordeinsxe2x80x9d, J. Inst. Brew., 103:297-306 (1997)). Mills et al. have reported the creation of a monoclonal library to beer proteins and polypeptides believed to be derived from the hordeins in malts (Mills et al., xe2x80x9cImmunological Study of Hydrophobic Polypeptides in Beerxe2x80x9d, J. Agric. Food Chem., 46:4475-4483 (1998)). Meanwhile, European Patent 0863153 by Ishibashi et al., and Kukai et al., xe2x80x9cDevelopment of Monoclonal Antibody Sandwich-ELISA for Determination of Beer Foam-Active Proteinsxe2x80x9d, J.Am. Soc. Brew. Chem., 56(2):43-46 (1998), both report the production and use of monoclonal antibodies in ELISA experiments directed against foam-active proteins having molecular weights between 40 and 50 kDa.
With respect to the lipid transfer proteins, only a single monoclonal antibody disclosed by Dickie has been reported (Dickie, xe2x80x9cImmunological Determination of Foam-Positive Hydrophobic Polypeptides in Barley and the Effects of Maltingxe2x80x9d, BRI Quarterly, 15-18 (October, 1997)). This antibody, identified as IFRN 1625, recognizes a ca. 8 kDa polypeptide found in Group 5 foam fractions, and is believed to be one of the proteins involved in lipid transfer. Dickie postulates that this 8 kDA polypeptide originates from LTP1 but presents no evidence to support this hypothesis.
What is needed is a set of monoclonal antibodies exhibiting specificity against lipid transfer proteins in either their native or modified forms. What is also needed is an assay capable of measuring and characterizing the content of native and denatured lipid transfer proteins in foaming beverages during various stages of their production process.
The present invention is summarized in that novel monoclonal antibodies against the native and denatured forms of barley lipid transfer protein 1 have been isolated.
The present invention includes monoclonal antibodies against the native form of barley lipid transfer protein 1 (LTP1). The first LTP1 antibody, identified as 3F7.1, has very strong reactivity to LTP1, no reactivity to fLTP and no reactivity to Protein. Z. Epitope mapping performed with 3F7.1 shows reactivity to amino acid sequences SEQ ID NO:14 and SEQ ID NO:15. The second LTP1 antibody, identified as 2C12.1 has very strong reactivity to LTP1, no reactivity to fLTP, and no reactivity to Protein Z. Epitope mapping performed with 2C12.1 shows reactivity to amino acid sequences SEQ ID NO:14 and SEQ ID NO:15, and a low level of reactivity to LTP1 amino acid sequences SEQ ID NO:10 and SEQ ID NO:11. The third LTP1 antibody, identified as 3G1.1, has strong reactivity to LTP1, no reactivity to fLTP, and no reactivity to Protein Z. Epitope mapping performed with 3G1.1 shows reactivity to amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
The present invention also includes monoclonal antibodies against the denatured form of barley lipid transfer protein 1 isolated from beer foam (fLTP). The first fLTP antibody, identified as 3D1.1, has strong reactivity to fLTP, some reactivity to LTP1, and no reactivity to Protein Z. Epitope mapping performed with 3D1.1 shows strong reactivity to amino acid sequences SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The second fLTP antibody, identified as 2E3.1 has strong reactivity to fLTP, no reactivity to LTP1, and some unconfirmed reactivity to Protein Z. Epitope mapping performed with 2E3.1 shows reactivity to amino acid sequences SEQ ID NO:16 and SEQ ID NO:17. The third fLTP antibody, identified as 3D11.1, has weak reactivity to fLTP, no reactivity to LTP1, and no reactivity to Protein Z. Epitope mapping performed with 3D11.1 shows reactivity to amino acid sequences SEQ ID NO:4 and SEQ ID NO:5, and a low level of reactivity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
In another embodiment, the present invention includes a kit comprising at least one LTP1 or fLTP antibody. In yet another embodiment, the present invention includes a kit comprising at least one LTP1 antibody and at least one fLTP antibody. In addition to the said antibodies, the kits may further comprise a 96-well plate, a sample-adsorbing buffer, a washing solution, a blocking solution, a substrate solution, a dilution of a secondary antibody and a calibration graph.
It is an object of the present invention to provide monoclonal antibodies useful in determining the content of both native lipid transfer proteins and denatured lipid transfer proteins in a foaming beverage during and after the beverage production process.
It is another object of the present invention to provide monoclonal antibodies that bind to native lipid transfer proteins and not denatured lipid transfer proteins.
It is another object of the present invention to provide monoclonal antibodies that bind to denatured lipid transfer proteins and not native lipid transfer proteins.
It is another object of the present invention to provide monoclonal antibodies that do not bind to protein Z.
It is one advantage of the present invention that foaming beverages can now be assayed to determine both the native lipid transfer protein content and the denatured lipid transfer protein content during various stages of the beverage production process.
Other objects, features and advantages of the present invention will become apparent after examination of the specification, claims and drawings.